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In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models

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They demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an allin-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9).

The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen.

Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials.

Authors: Chaoran Yin, Ting Zhang, Xiying Qu, Yonggang Zhang, Raj Putatunda, Xiao Xiao, Fang Li, Weidong Xiao, Huaqing Zhao, Shen Dai, Xuebin Qin, Xianming Mo, Won-Bin Young, Kamel Khalili, and Wenhui Hu

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Point-of-care mobile digital microscopy for the most common helminth eggs

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MoMic digital microscope scanner (1) with external motor unit attached (2). The microscope glass (3) is placed in the slide holder (4), which is placed in the microscope and navigated from the motor unit. The device is connected to and operated from a laptop computer (5) running software (6) for operation of the device.

Microscopy remains the gold standard in the diagnosis of neglected tropical diseases. As resource limited, rural areas often lack laboratory equipment and trained personnel, new diagnostic techniques are needed. Low-cost, point-of-care imaging devices show potential in the diagnosis of these diseases. Novel, digital image analysis algorithms can be utilized to automate sample analysis.

Authors: Oscar Holmström , Nina Linder, Billy Ngasala, Andreas Mårtensson, Ewert
Linder, Mikael Lundin, Hannu Moilanen, Antti Suutala, Vinod Diwan & Johan
Lundin

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The use of error and uncertainty methods in the medical laboratory

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Error methods – compared with uncertainty methods – offer simpler, more intuitive and practical procedures for calculating measurement uncertainty and conducting quality assurance in laboratory medicine.

However, uncertainty methods are preferred in other fields of science as reflected by the guide to the expression of uncertainty in measurement. When laboratory results are used for supporting medical diagnoses, the total uncertainty consists only partially of analytical variation. Biological variation, pre- and postanalytical variation all need to be included. Furthermore, all components of the measuring procedure need to be taken into account.

Performance specifications for diagnostic tests should include the diagnostic uncertainty of the entire testing process. Uncertainty methods may be particularly useful for this purpose but have yet to show their strength in laboratory medicine. The purpose of this paper is to elucidate the pros and cons of error and uncertainty methods as groundwork for future consensus on their use in practical performance specifications. Error and uncertainty methods are complementary when evaluating measurement data.

Authors: Wytze P. Oosterhuis, Hassan Bayat, David Armbruster, Abdurrahman Coskun,
Kathleen P. Freeman, Anders Kallner, David Koch, Finlay Mackenzie, Gabriel Migliarino,
Matthias Orth, Sverre Sandberg, Marit S. Sylte, Sten Westgard and Elvar Theodorsson

Published Online: 2017-08-10 | DOI: https://doi.org/10.1515/cclm-2017-0341

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Metrological traceability of test results

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Metrological traceability is a 10-syllable term that is difficult to say, but important to understand. It is essential to the validity of clinical results, so it must always be considered. If clinical test results are to be comparable across time, across different instrument platforms, or across labs, they must be metrologically traceable to the same measurement reference.

Establishing metrological traceability requires that the calibration material used for a result (or a run, or series of calibration materials), has certified property values that have an unbroken chain of comparisons going back to the appropriate national or international standard—for example, the use of certfied reference materials (CRM) from a National Metrology Institute (NMI) such as the National Institute of Standards and Technology (NIST) which have assigned values that are traceable to the International System of units (SI units). Other respected appropriate standards that are used to establish metrological traceability and are common to the clinical lab industry include those produced by the World Health Organization (WHO) with arbitrary units, often called “International Units” (IU).

Metrological traceability is the subject of several ongoing efforts at the Joint Committee for Traceability in Laboratory Medicine (JCTLM), the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC), and the American Association for Clinical Chemistry (AACC) Harmonization Task Force and the International Consortium for Harmonization. It has been addressed by the International Organization for Standardization (ISO), by the Clinical and Laboratory Standards Institute (CLSI), and by the International Cooperation for Traceability in Analytical Chemistry (CITAC).

The JCTLM is a project of the International Bureau of Pounds and Measures (BIPM), and is composed of representatives from BIPM, IFCC, and the International Laboratory Accreditation Cooperation (ILAC). ILAC is a cooperation of 95 laboratory accreditation bodies from 80 countries that accredit testing and calibration laboratories to the requirements of ISO/IEC 17025 and ISO 15189.

The formal definition

As defined by the International Vocabulary of Metrology (VIM), metrological traceability is a “property of the measurement result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty.” Notes in the definition clarify that a “reference” can be a definition of a measurement unit, a measurement procedure, or a measurement standard, and (for internationally recognized accreditation) the reference should be a national or international reference standard with traceability to the International System of Units (SI), where available.

It is important to recognize that metrological traceability is different from other forms of “traceability” within a laboratory. It is different from the traceability of a sample as it moves through the measurement system (sometimes called “chain of custody”), and it is different from the “traceability of calculations,” which involve tracking original instrument indications and any manual calculations or transfer of results. Metrological traceability relates to the measurement result itself, the data that is produced in the laboratory that is provided to the “customer,” and is used to make critical decisions that could have direct impact on the health of a patient. Metrological traceability is about the units of measurement, how the measurement results compare to other measurement results, and what the measurement results mean. Metrological traceability helps ensure that we are comparing apples to apples and, furthermore, that we are comparing a Gala apple to a Gala apple—and with what level of certainty we can say that our Gala apple is indeed a Gala apple. Metrological traceability should not be confused with tracking the sample or the result as they move through the laboratory; rather, it relates a measurement result back to a reference that allows (or not) comparison to other results for the same analyte.

Achieving metrological traceability

Here are three common examples of how metrological traceability can be achieved:

  1. Calibration to the SI (or other reference) through a primary reference material from an NMI (for example, in the United States, to certified property values contained in SRMs from NIST). This can be conducted by a single laboratory (for example, glucose in serum).
  2. Use of a reference measurement method by a network of expert laboratories using a well-described reference measurement method. This requires that all laboratories are independently verified to be competent (for example, creatinine in serum).
  3. Use of a reference method by a single expert laboratory (for example, total cholesterol in serum).

Establishing metrological traceability can be expensive, but past efforts have demonstrated benefits to anyone who has tracked interlaboratory agreement of measurements in recent years; that is, whether improvements in method agreement for creatinine, cholesterol, or glucose seem like recent improvements to you (or, in the experience of the principal author, what has occurred with the use of beta hCG measurements in the past 35 years). Harmonization of results obtained by different measurement methods is possible for every analyte.

If you follow rules that require strict adherence to the manufacturers’ guidelines, then there is little you can do to affect the traceability of results that you report. We recommend contacting the manufacturers and requesting information detailing how they ensure metrological traceability of results using their equipment. If you have inter-method agreement factors to harmonize results from different instruments in your laboratory, there is also little else that you can do to improve traceability.

Remember that metrological traceability requires measurement results to be traceable to SI units or another appropriate reference when that isn’t possible. In a clinical laboratory, metrological traceability is often achieved through the use of certified reference materials or the use of reference methods. The JCTLM maintains a searchable database of certified reference materials, accepted reference methods, and standardization organizations, available at this address: http://www.bipm.org/jctlm/.

Source: MLO

Eliminating creatine kinase–myocardial band testing in suspected acute coronary syndrome

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Cardiac biomarker testing is estimated to occur in nearly 30 million emergency department visits nationwide each year in the United States. The American College of Cardiology/European Society of Cardiology indicate that cardiac troponin is the biomarker of choice owing to its nearly absolute myocardial tissue specificity and high clinical sensitivity for myocardial injury. Multiple academic medical centers have implemented interventions to eliminate the routine ordering of creatine kinase–myocardial band tests, with published patient safety outcomes data; however, creatine kinase–myocardial band testing is still ordered in many hospitals and emergency departments. Eliminating a simple laboratory test that provides no incremental value to patient care can lead to millions of health care dollars saved without adversely affecting patient care quality, and in this case potentially improving patient care.

Introduction

Acute coronary syndrome (ACS) is one of the leading causes of mortality in the United States. Since the 2000 American College of Cardiology (ACC)/European Society of Cardiology (ESC) redefinition of acute myocardial infarction (AMI), which was revised into the 2007 and 2012 Universal Definition of AMI, cardiac troponin (cTn) has been the biomarker of choice owing to its nearly absolute myocardial tissue specificity and high clinical sensitivity for myocardial injury.1,2 Cardiac troponin is preferred by both clinical (ACC/ESC/American Heart Association [AHA])3- 5 and biochemical (National Academy of Clinical Biochemistry)6,7 guideline groups, for similar reasons. In the most recent universal definition of AMI, creatine kinase-myocardial band (CK-MB) is described as an alternative to be used only if cTn is not available.2 The 2014 AHA/ACC guidelines conclude that CK-MB provides no additional value for diagnosing AMI (class III, level of evidence A).5

The ideal biomarker test for the diagnosis of AMI should be highly sensitive and specific, rapidly obtained and analyzed, and lead to treatment decisions that provide high value, in terms of clinical benefit relative to cost.8 The importance of this goal stems from the sheer number of patients receiving cardiac biomarker testing and the resulting contribution to health care expenditure in the United States each year. In 2010, Makam and Nguyen9 showed via the National Hospital Ambulatory Medical Care Survey (n = 44 448 emergency department [ED] visits) that cardiac biomarker testing (both cTn and CK-MB) occurred in 16.9% of all visits to the ED. The authors extrapolated this to represent 28.6 million ED visits nationwide. Considering Medicare’s 2016 Clinical Diagnostic Laboratory Fee Schedule10 (national payment amounts for cTn and CK-MB of $13.40 and $15.73, respectively), approximately $416 million is spent on cardiac biomarker testing annually. Not included in these estimates are additional unnecessary expenditure and potential harm associated with subsequent noninvasive and invasive testing that may result from false-positive results.

Once the cornerstone of AMI diagnosis, CK-MB has not yet been eliminated from practice despite considerable evidence supporting cTn as the preferred biomarker.8,11 Data published after distribution of the ACC/ESC/AHA3- 5 recommendations show the these clinical practice guidelines have not succeeded in refining practice. Specifically, CK-MB is still used in many US clinical pathology laboratories and US EDs.12,13 Based on the College of American Pathologists proficiency survey in 2013, 1558 of 1995 national US laboratories (77%) still use CK-MB.12 In 2009, Parker and Suter13 surveyed 98 ED physician leaders in 21 academic and 77 community EDs regarding the use of cardiac biomarkers and found that 77% (76 of 98) still use CK-MB. Collinson et al14 found that of the nearly 40% of North American and European laboratories (n = 533) offering CK-MB testing in 2006, 25% continued to offer the test even after the national guidelines discussed above recommended against its use. Cappelletti et al15 added to the findings by Collinson et al14 by adding data from Italy (n = 126 laboratories) that showed 20.2% of laboratories continue to use CK-MB.

This retention of CK-MB has been attributed to clinicians’ reluctance to rely on cTn in certain clinical situations, as well as clinician familiarity.8,11 In 2010, a study coauthored by 7 large academic medical centers labeled CK-MB as 1 of 10 tests that no longer provide value,16 and a growing number of medical centers have abandoned use of CK-MB.8 With the goal of advancing high value practice, as defined by the ACC, ESC, AHA3- 5 and National Academy of Clinical Biochemistry, this implementation guideline is designed to assist institutions in eliminating unnecessary CK-MB testing.

Evidence-Based Guidelines: Troponin and CK-MB Tests

Numerous studies have compared CK-MB and cTn with respect to their diagnostic precision related to the initial diagnosis of AMI, the assessment of reinfarction, and prognosis after major cardiovascular events. Cardiac troponin has been shown to be highly sensitive for AMI (99.2%)17 and to be more specific than CK-MB (lower false-positive rate) owing to its exclusivity in cardiac myocytes (vs CK-MB, which may be elevated with skeletal muscle damage).16- 19 Hawkins et al20 calculated a specificity of cTn testing as high as 92% compared with 40% for CK-MB. Lin et al21 observed that among patients with suspected AMI who also had negative cTn results, approximately 10% of patients had abnormally high CK-MB.22 Similarly, Antman et al23 evaluated cardiac biomarker levels in patients with ACS symptoms and found that 238 of 948 patients (25%) initially classified as having unstable angina owing to normal CK-MB tests actually had elevated cTn levels. Thus, the use of CK-MB may potentially misclassify individuals with ACS by mistakenly diagnosing unstable angina in some patients with true myocardial infarction.

In patients with chronic renal disease, the diagnosis of ACS can be challenging, as cTn levels may be chronically elevated. However, CK-MB has been shown to provide no incremental value over cTn in the diagnosis of ACS in patients with chronic renal disease.24,25 In fact, CK-MB levels are often elevated in patients on dialysis in the absence of ACS signs and/or symptoms.25

In terms of time to diagnosis, both CK-MB and troponin are equally rapid in ACS diagnosis,8 but with more sensitive assays, cTn rises much more rapidly.26- 28 In addition to its superior sensitivity and specificity, cTn yields stronger prognostic information. In patients with ACS, the CRUSADE investigators (n = 29 357)29 showed that elevated cTn is associated with in-hospital mortality, regardless of CK-MB levels. However, when CK-MB levels are elevated in the presence of a normal cTn, in-hospital mortality is comparable to CK-MB levels and cTn both being negative.29- 31 Most important, those with elevated CK-MB values but normal cTn levels do not manifest an adverse prognosis.29- 31

Despite a longstanding, commonly held physician belief that CK-MB is more useful than cTn for detecting reinfarction, no study has shown that CK-MB levels are superior to cTn in this regard.32,33 Indeed, most data confirm that cTn provides far better estimates because it is less impacted by changes in the amount of marker released with reperfusion.34 Single values correlate closely with infarct size as determined by cardiac magnetic resonance imaging.35 Apple et al36 showed that both biomarker levels rise similarly with reinfarction. The most recent ACC/AHA/ESC guidelines support the use of cTn over CK-MB for diagnosing reinfarction.3- 5

Most patients with ACS are treated with either surgical or percutaneous revascularization.37,38 The use of CK-MB or cTn following percutaneous coronary intervention (PCI) is controversial, as there is no current consensus regarding the definition, prognosis, and subsequent treatment of periprocedural myocardial infarction diagnosed by cardiac biomarker elevation alone. Some groups have suggested that CK-MB is superior to cTn in terms of detecting periprocedural myocardial infarction,38 while others have argued that post-PCI cardiac biomarkers are not useful because prognosis is related to the pre-PCI cTn value, and when that is taken into account, post-PCI myocardial injury is not an important clinical end point.39 Even if clinicians decide to assess cardiac biomarkers in this setting, post-PCI cTn and CK-MB values provide equally accurate prognostic information.39 Because this guideline is focused on cardiac biomarker use in routine ACS diagnosis, rather than cardiac biomarker use following PCI, we defer further discussion and/or recommendation with respect to this ongoing clinical controversy.

Another potential area of controversy concerns the appropriate use of cTn in clinical practice.40 The major strength of using cTn—its specificity for cardiac injury—is also a potential shortcoming, because cTn detects myocardial injury irrespective of the specific cause. Consequently, many noncardiac conditions associated with secondary myocardial injury, such as pulmonary embolus, severe anemia, and severe hypotension from any cause, are associated with abnormal cTn. Wilson et al40 reported a high prevalence of elevated cTn in a large group of hospitalized patients due to noncardiac conditions and has suggested that this may represent an example of overuse of cTn. Another group has reported a small series of patients in which the quantity of troponins ordered per patient exceeded guideline recommendations.41 We acknowledge that the real-world use of cTn, with respect to appropriate clinical indication and quantity over time, deserves further study and quality improvement initiatives designed to refine usage.

Finally, Saenger et al8 have observed that concomitant use of CK-MB and cTn is often confusing for physicians, and they are aware of situations in which such confusion has negatively affected patient care. An example of a potentially confusing clinical situation is when a patient is admitted with possible symptoms of ACS and is found to have an abnormally elevated CK-MB levels in the setting of sequentially normal troponin levels. Although normal sequential troponin values exclude the diagnosis of acute myocardial injury—and are associated with a favorable short-term prognosis regardless of the CK-MB levels—some physicians might erroneously conclude that the patient has suffered acute myocardial injury due to CK-MB elevation. Others might wonder if there is another condition (ie, skeletal muscle breakdown) responsible for the CK-MB elevation and might erroneously devalue the importance of the patient’s symptoms and electrocardiogram in making the diagnosis of ACS.42 We would argue that elimination of routine CK-MB ordering is not only high value because it offers no benefit and results in considerable cost but also because elimination of CK-MB may reduce physician confusion, improve understanding of the proper use of cTn, and consequently reduce potential patient harm.

Safety and Quality Outcomes Data: Eliminating CK-MB Testing

Multiple academic medical centers have implemented interventions to eliminate the routine ordering of CK-MB and measured associated cost savings and impact on patient safety (Table).12,43- 46 Larochelle et al44 recorded data on patients with suspected ACS (n = 60 494 patients preintervention; n = 24 341 patients postintervention) over a period of 43 months (31 months preintervention; 12 months postintervention). They initially developed an institutional guideline in collaboration with cardiologists to specify appropriate ordering of cardiac biomarkers for the diagnosis of AMI. The guideline advised practitioners to order cTn alone, without CK-MB. The authors then implemented multiple interventions, including educational sessions for internal medicine and ED physicians, dissemination of pocket-sized quick reference cards with the recommended ordering algorithm, and removal of CK-MB from ACS routine order sets within the computerized provider order entry (CPOE) system. A Best Practice Advisory (BPA) was created in the CPOE system using the institutional guideline to alert physicians who attempted to order CK-MB. Physicians could, if desired, order CK-MB manually. By 12 months postintervention, the estimated number of CK-MB tests per patient was 0, representing an absolute change of −0.96 CK-MB tests per patient (95% CI, −1.00 to −0.92). The authors estimated a 95% reduction in CK-MB tests, translating to $720 000 in annual savings.

Similarly, Baron et al43 removed CK-MB from routine order sets and implemented a BPA into their CPOE system without additional educational sessions. After only 2 months postimplementation, they noted an 87% decrease in CK-MB orders. In addition, they noted fewer orders after searches and fewer searches for CK-MB over this time, arguing for a long-term educational benefit for the BPA. Multiple other groups, including Mayo Clinic, simply removed CK-MB from routine order sets and found 80.0% to 99.8% reductions in CK-MB orders with significant cost savings and without negative impact on patient care or missed AMI diagnoses.8,12,43- 46

Implementation Blueprint: Eliminating CK-MB Testing

With clear evidence to support elimination of CK-MB from clinical care for the diagnosis of ACS and based on experience eliminating CK-MB at our respective institutions, we devised a blueprint for other institutions to use in enacting quality improvement initiatives. The methodology and theory of the blueprint and quality improvement initiative are based on the US Health Resources and Services Administration strategies for developing and implementing a quality improvement initiative, with a focus on education, action, and measurement of results.47 The leader of each institution should assess applicability of this blueprint based on the CK-MB ordering frequency at their respective institution.

  1. Design and implement a hospital-wide educational campaign.
    • Prior to implementation, it is important for health care leaders to establish sufficient organizational readiness for change, specifically in conjunction with these stakeholders, whose ordering practices will be affected by such changes. Tools to measure readiness for change have been previously studied and may be used.48,49
    • Collaborate with physician representatives from the departments of cardiology, internal medicine, and emergency medicine. Frontline physicians in these departments order the majority of cardiac biomarkers within most health care institutions and are the primary stakeholders in the current system of care for ACS patients.
    • Academic institutions must engage the house staff as members of the quality improvement team for an effective initiative.
    • Collaborate with secondary stakeholders, such as pathology and/or laboratory staff, to acquire insight and advice on these changes.
    • Inform physicians that CK-MB adds to the health care system financial burden without adding value to patient care.
    • Present the evidence supporting elimination of CK-MB and exclusive use of cTn to diagnose AMI, identify reinfarction, and estimate infarct size. Education may be provided through various venues, including lectures, pocket cards (Figure 2), online modules, social media demonstrations, and simulations.44
  2. Partner with information technology and/or laboratory medicine staff to remove CK-MB from standardized ACS routine order sets. Doing this simple step alone has been shown to significantly reduce CK-MB ordering (Table).8,12,45,46
  3. Partner with information technology and/or laboratory medicine staff to create and integrate a best practice alert into the CPOE to appear when clinicians order CK-MB, such as:
    • According to national guidelines, troponin is the preferred biomarker for detecting myocardial injury; CK-MB is only appropriate if troponin testing is unavailable. Figure 1 provides an example.43,44
  4. Measure data preintervention and postintervention (efficacy points).
    • Number of cTn and CK-MB tests ordered, including stratification by department and patient setting (ED, inpatient, medicine vs nonmedicine units, ICU vs non-ICU).
    • Incidence, missed diagnoses, and mortality of AMI to ensure patient safety.
    • Review cases where CK-MB is still ordered to determine if it provides value.
    • If necessary, track usage by physician to develop performance feedback profiles.
    • Calculate reduction in charges to patients and health plans, as well as any decrease in hospital costs.

Though seemingly straightforward to articulate, we acknowledge that significant barriers to implementation exist, and in this case the biggest hurdle has been convincing physicians who have ordered CK-MB for years to change their practice. Successful deimplementation of CK-MB requires leadership support, education, and reassurance that diagnostic efficacy will not be compromised. Other challenges relate to manpower as well as modifications and data collection from the electronic medical record. To optimize manpower, academic centers can engage house staff because this will also ensure longstanding change. Again, leadership commitment to allocating information technology resources for data collection and clinical decision support tools is critical.

Conclusions

Creatine kinase–myocardial band testing provides no incremental value to patient care, and its elimination can lead to millions of health care dollars saved without adversely affecting patient care. This is backed by both strong evidence-based guidelines and experiences from multiple institutions. The blueprint presented here should be carefully considered by health care leaders and clinicians as the first step to finally putting the CK-MB laboratory test to rest.

Acknowledgements: This article is the first collaborative scholarly activity produced by faculty in the High Value Practice Academic Alliance (HVPAA). The HVPAA organization includes faculty from dx.doi.org/…161/CIRCULATIONAHA.107.187397more than 80 academic medical centers collaborating on quality improvement, research, and education related to high-value healthcare.

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19. Adams  JE  III, Bodor  GS, Dávila-Román  VG,  et al.  Cardiac troponin I. A marker with high specificity for cardiac injury.  Circulation. 1993;88(1):101-106.
20. Hawkins  RC, Tan  HL.  Comparison of the diagnostic utility of CK, CK-MB (activity and mass), troponin T and troponin I in patients with suspected acute myocardial infarction.  Singapore Med J. 1999;40(11):680-684.
21. Lin  JC, Apple  FS, Murakami  MM, Luepker  RV.  Rates of positive cardiac troponin I and creatine kinase MB mass among patients hospitalized for suspected acute coronary syndromes.  Clin Chem. 2004;50(2):333-338.
22. Adams  JE  III, Abendschein  DR, Jaffe  AS.  Biochemical markers of myocardial injury. Is MB creatine kinase the choice for the 1990s?  Circulation. 1993;88(2):750-763.
23. Antman  EM, Tanasijevic  MJ, Thompson  B,  et al.  Cardiac-specific troponin I levels to predict the risk of mortality in patients with acute coronary syndromes.  N Engl J Med. 1996;335(18):1342-1349.
24. Flores-Solís  LM, Hernández-Domínguez  JL.  Cardiac troponin I in patients with chronic kidney disease stage 3 to 5 in conditions other than acute coronary syndrome.  Clin Lab. 2014;60(2):281-290.
25. Jaffe  AS, Ritter  C, Meltzer  V, Harter  H, Roberts  R.  Unmasking artifactual increases in creatine kinase isoenzymes in patients with renal failure.  J Lab Clin Med. 1984;104(2):193-202.
26. Eggers  KM, Oldgren  J, Nordenskjöld  A, Lindahl  B.  Diagnostic value of serial measurement of cardiac markers in patients with chest pain: limited value of adding myoglobin to troponin I for exclusion of myocardial infarction.  Am Heart J. 2004;148(4):574-581.
27. Ilva  T, Eriksson  S, Lund  J,  et al.  Improved early risk stratification and diagnosis of myocardial infarction, using a novel troponin I assay concept.  Eur J Clin Invest. 2005;35(2):112-116.
28. Kavsak  PA, MacRae  AR, Newman  AM,  et al.  Effects of contemporary troponin assay sensitivity on the utility of the early markers myoglobin and CKMB isoforms in evaluating patients with possible acute myocardial infarction.  Clin Chim Acta. 2007;380(1-2):213-216.
29. Newby  LK, Roe  MT, Chen  AY,  et al; CRUSADE Investigators.  Frequency and clinical implications of discordant creatine kinase-MB and troponin measurements in acute coronary syndromes.  J Am Coll Cardiol. 2006;47(2):312-318.
30. Goodman  SG, Steg  PG, Eagle  KA,  et al; GRACE Investigators.  The diagnostic and prognostic impact of the redefinition of acute myocardial infarction: lessons from the Global Registry of Acute Coronary Events (GRACE).  Am Heart J. 2006;151(3):654-660.
31. Storrow  AB, Lindsell  CJ, Han  JH,  et al; EMCREG-i*trACS Investigators.  Discordant cardiac biomarkers: frequency and outcomes in emergency department patients with chest pain.  Ann Emerg Med. 2006;48(6):660-665.
32. Panteghini  M, Cuccia  C, Bonetti  G, Giubbini  R, Pagani  F, Bonini  E.  Single-point cardiac troponin T at coronary care unit discharge after myocardial infarction correlates with infarct size and ejection fraction.  Clin Chem. 2002;48(9):1432-1436.
33. Licka  M, Zimmermann  R, Zehelein  J, Dengler  TJ, Katus  HA, Kübler  W.  Troponin T concentrations 72 hours after myocardial infarction as a serological estimate of infarct size.  Heart. 2002;87(6):520-524.
34. Gibbons  RJ, Valeti  US, Araoz  PA, Jaffe  AS.  The quantification of infarct size.  J Am Coll Cardiol. 2004;44(8):1533-1542.
35. Giannitsis  E, Steen  H, Kurz  K,  et al.  Cardiac magnetic resonance imaging study for quantification of infarct size comparing directly serial versus single time-point measurements of cardiac troponin T.  J Am Coll Cardiol. 2008;51(3):307-314.
36. Apple  FS, Murakami  MM.  Cardiac troponin and creatine kinase MB monitoring during in-hospital myocardial reinfarction.  Clin Chem. 2005;51(2):460-463.
37. Cannon  CP, Weintraub  WS, Demopoulos  LA,  et al; TACTICS (Treat Angina with Aggrastat and Determine Cost of Therapy with an Invasive or Conservative Strategy)—Thrombolysis in Myocardial Infarction 18 Investigators.  Comparison of early invasive and conservative strategies in patients with unstable coronary syndromes treated with the glycoprotein IIb/IIIa inhibitor tirofiban.  N Engl J Med. 2001;344(25):1879-1887.
38. Grines  CL, Dixon  S.  A nail in the coffin of troponin measurements after percutaneous coronary intervention.  J Am Coll Cardiol. 2011;57(6):662-663.
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40. Wilson  G, Barkley  K, Slicker  K, Kowal  R, Pope  B, Michel  J.  Overuse of troponin? a comprehensive evaluation of testing in a large hospital system.  J Hosp Med. 2017;12(5):329-331.
41. Fraga  OR, Sandoval  Y, Love  SA,  et al.  Cardiac troponin testing is overused after the rule-in or rule-out of myocardial infarction.  Clin Chem. 2015;61(2):436-438.
42. Pierce  GF, Jaffe  AS.  Increased creatine kinase MB in the absence of acute myocardial infarction.  Clin Chem. 1986;32(11):2044-2051.
43. Baron  JM, Lewandrowski  KB, Kamis  IK, Singh  B, Belkziz  SM, Dighe  AS.  A novel strategy for evaluating the effects of an electronic test ordering alert message: optimizing cardiac marker use.  J Pathol Inform. 2012;3:3.
44. Larochelle  MR, Knight  AM, Pantle  H, Riedel  S, Trost  JC.  Reducing excess cardiac biomarker testing at an academic medical center.  J Gen Intern Med. 2014;29(11):1468-1474.
45. Le  RD, Kosowsky  JM, Landman  AB, Bixho  I, Melanson  SE, Tanasijevic  MJ.  Clinical and financial impact of removing creatine kinase-MB from the routine testing menu in the emergency setting.  Am J Emerg Med. 2015;33(1):72-75.
46. Sullivan  P, Waymack  J, Griffen  D, Jaeger  C.  Effectively reducing CK-MB utilization using computer order entry in the emergency department.  Am J Med Qual. 2017;32(1):107.
47. Health Resources and Services Administration. Quality Improvement. https://www.hrsa.gov/quality/toolbox/methodology/qualityimprovement/. Accessed May 12, 2017.
48. Shea  CM, Jacobs  SR, Esserman  DA, Bruce  K, Weiner  BJ.  Organizational readiness for implementing change: a psychometric assessment of a new measure.  Implement Sci. 2014;9:7
49. Helfrich  CD, Li  YF, Sharp  ND, Sales  AE.  Organizational readiness to change assessment (ORCA): development of an instrument based on the Promoting Action on Research in Health Services (PARIHS) framework.  Implement Sci. 2009;4:38.
Authors: Matthew D. Alvin, MD, MBA, MS, MA1; Allan S. Jaffe, MD2; Roy C. Ziegelstein, MD, MACP3; et al Jeffrey C. Trost, MD3
  1. Department of Radiology and Radiological Sciences, Johns Hopkins Hospital, Baltimore, Maryland
  2. Division of Cardiology, Department of Medicine, Mayo Clinic, Rochester, Minnesota
  3. Division of Cardiology, Department of Medicine, Johns Hopkins Bayview Medical Center, Baltimore, Maryland

Source: jamanetwork.com

Gut viruses tied to potentially deadly complication of bone marrow transplant

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GvHD affects up to 60 percent of patients who undergo bone marrow stem-cell transplants, and kills about half of those affected. After transplants, to prevent a recipient’s immune cells from laying siege to unfamiliar donor cells and rejecting them, clinicians often use drugs to suppress the immune response. GvHD is a mirror image of organ rejection, in which immune cells in the transplant attack its new host, the patient.

Despite the pervasiveness of this disease, there isn’t yet a clear way of foretelling patients’ risk of developing it before they go into surgery. The new study, published online July 31, 2017, in Nature Medicine, unveils a viral biomarker that could allow clinicians to assess patients’ risk of an acute form of the disease known as enteric GvHD, which affects the gastrointenstinal system.

The team used a technique known as metagenomic next-generation sequencing (mNGS) – which can rapidly and concurrently sequence genetic material of all organisms present in any biological sample – to catalog microbes in patients’ digestive tracts, monitoring the evolving bacterial and viral population throughout the transplantation process.

Although mNGS analyses of bacterial populations, called microbiomes, have been much in the news, fewer studies have focused on “viromes,” the term for viral populations.

“Viromes can play an important part in health and disease,” said Charles Chiu, MD, PhD, an associate professor of laboratory medicine at UCSF and principal investigator of the study. “Our goal was to understand what impact transplantation has on the gut virome.”

In the new work, the researchers scanned stool samples taken from 44 patients before they received a transplant and up to six weeks after, and sequenced all the DNA and RNA in the samples in order to assemble a roster of their microbial passengers.

Using this technique, the researchers identified a number of viruses that flared up in the guts of patients who developed the deadly condition. Of particular note were members of the picobirnavirus (PBV) family: the presence of these viruses before transplantation, even in very small populations, was a reliable sign that a patient would likely develop the disease after a transplant.

“I would’ve expected herpesviruses or adenoviruses to be the more likely cause of infection,” said Chiu. “We wouldn’t have picked up picobirnaviruses were it not for the metagenomics approach.”

PBVs are a very diverse family of viruses – more diverse than HIV, said Jérôme Le Goff, PhD, associate professor at the University of Paris Diderot and lead author of the new study. “It’s very difficult to design a single test to detect all viruses simultaneously,” said Le Goff. “So for many years, labs did not have the means to look for PBV.” Indeed, each of the 18 patients who tested positive for PBV was carrying a different strain, a diversity that makes it challenging to detect PBVs using a simple lab test.

The team also observed a previously unreported “bloom” of other resident viruses in patients that occurred three to five weeks after they had received transplants. Intriguingly, the onset of GvHD appeared to trigger the late awakening of these covert viruses, laying to rest a longstanding chicken-and-egg debate: which comes first, viral infection or GvHD? The researchers conclude that much of the viral flare they saw is due to reactivation of latent gut infections following transplantation.

Given the potential utility of PBV as a predictive biomarker, Chiu and his team now hope to develop a metagenomics-based test to screen patients before transplantation. “We also saw shifts in the microbiome but those in the virome were more pronounced,” said Chiu. “Loss of bacteria colonizing the gut has been thought to predispose patients to GvHD; here we show that shifts in the virome may also play a role in the occurrence of this disease.”

Although the new study strongly implicates PBVs in the onset of GvHD, it is too early to tell whether or how these viruses trigger the disease. The team is now enrolling more adult and pediatric patients – both in Paris and at UCSF – to expand their analyses and uncover the mechanism by which the virus modulates the risk of disease. A systematic understanding of the virus’s role could ultimately inform whether using antiviral drugs or tweaking the body’s immune response would be the best strategy to temper the disease.

“It would be great to have a tool that can be used to assess GvHD risk in these patients before they undergo a transplant,” Chiu said, a step that Le Goff said could lead to new therapies. “We hope that in the next few years we will find a way to prevent -associated GvHD,” said Le Goff.

Source: medicalxpress.com

Discovery of new prostate cancer biomarkers could improve precision therapy

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In the publication, the authors explain the role of mutations within the SPOP gene on the development of resistance to one class of drugs. SPOP mutations are the most frequent genetic changes seen in primary prostate cancer. These mutations play a central role in the development of resistance to drugs called BET-inhibitors.

BET, bromodomain and extra-terminal domain, inhibitors are drugs that prevent the action of BET proteins. These proteins help guide the abnormal growth of cancer cells.

As a therapy, BET-inhibitors are promising, but drug resistance often develops, says Haojie Huang Ph.D., senior author and a molecular biologist within Mayo Clinic’s Center for Biomedical Discovery. Prostate cancer is among the most diagnosed malignancies in the United States. It is also the third leading cause of cancer death in American men, according to the American Cancer Society. Because of this, says Dr. Huang, improving treatments for prostate cancer is an important public health goal.

In the publication, the authors report SPOP mutations stabilize BET proteins against the action of BET-inhibitors. By this action, the mutations also promote cancer cell proliferation, invasion and survival.

“These findings have important implications for prostate cancer treatment, because SPOP mutation or elevated BET protein expression can now be used as biomarkers to improve outcome of BET inhibitor-oriented therapy of prostate cancer with SPOP mutation or BET protein overexpression,” says Dr. Huang.

Mutations in the SPOP gene (depicted on the left, below) can then be used to guide administration of anti-cancer drugs (labeled treatment A through D below) in patients with prostate cancer:

The Nature Medicine publication presents four major discoveries:

  • BET proteins (BRD2, BRD3 and BRD4) are true degradation substrates of SPOP.
  • SPOP mutations cause elevation of BET proteins in prostate cancer patient specimens.
  • Expression of SPOP mutants leads to BET-inhibitor resistance and activation the AKT-mTORC1 pathway that promotes cancerous cell growth and survival.
  • Co-administration of AKT inhibitors overcomes BET inhibitor resistance in SPOP-mutated prostate cancer.

Mayo Clinic Ventures, the technology commercialization arm of Mayo Clinic, has a patent application in place for this promising prostate cancer biomarker and therapeutic technology.

In addition to Dr. Huang, other authors from Mayo Clinic are: Dejie Wang, Ph.D.;Yu Zhao, Ph.D.; Zhenqing Ye, Ph.D.; Yuqian Yan, Ph.D.; Yinhui Yang, M.D.; Di Wu, Ph.D.; Yundong He, Ph.D.; Jun Zhang, M.D.; Liguo Wang, Ph.D.

Authors from Fudan University, Nanchang University, Xinhua Hospital at Shanghai Jiao Tong University Medical School, and Second Military Medical University in China are listed in the publication.

The authors report no conflicts of interest. Funding for this work was provided by the National Institutes of Health, the U.S. Department of Defense, the National Natural Science Foundation of China, and the National Key Research and Development Plan of China ─ Precision Medicine Project.

Source: newsnetwork.mayoclinic.org

Research That Could Significantly Improve Treatment for Autism Unveiled at 69th AACC Annual Scientific Meeting

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The research, revealed at the 69th AACC Annual Scientific Meeting & Clinical Lab Expo in San Diego, could lead to promising new treatment options for children with autism as well as earlier detection of the disorder.

The scientific community has made great strides in understanding the genetic and environmental risk factors behind autism spectrum disorder, but the core mechanisms of the condition remain a mystery. Although different forms of therapy can improve autism symptoms, researchers have yet to uncover a comprehensive treatment solution. In addition, autism is difficult to diagnose before the second year of life. An assay that detects autism earlier in children or newborns could lead to more effective therapeutic and medicinal interventions. Recent research suggests that an individual’s metabolism and gut microbiome—the ecosystem of microorganisms in the digestive system—play a pivotal role in the development of autism spectrum disorder and could yield answers to address these issues.

A research team led by Antonio Noto, PhD, of the department of surgical sciences at the University of Cagliari in Cagliari, Italy, set out to elucidate the role of metabolism and the gut microbiome in children with autism. The team took urine and stool samples from 21 autistic patients from 4–16 years old and 21 non-affected siblings from 4–17 years old. They analyzed the urine samples with proton nuclear magnetic resonance spectroscopy to measure the amounts of metabolites present. Noto’s team also performed targeted sequencing of 16S bacterial ribosomal RNA genes in the stool samples to profile the children’s gut microorganisms.

The researchers found that urinary metabolites in autism spectrum disorder patients were markedly different from those in the non-affected siblings. Autism patients showed signs of an imbalance in amino acid metabolism, with increased levels of hippurate, glycine, creatine, tryptophan, and D-threitol and decreased levels of glutamate, creatinine, lactate, valine, betaine, and taurine. Additionally, autism patients exhibited gut dysbiosis—they had a significant increase in the presence of Clostridium bacterial species compared with the non-affected siblings.

The findings raise the possibility of developing new treatment methods for autism in newborns based on correcting gut microorganism imbalances through diet modification or fecal transplantation. “This is very important because gut dysbiosis alters the permeability of the intestinal mucosa and is the origin of abnormal metabolites which can easily pass into circulation and reach the brain,” said Michele Mussap, MD, a co-author of the study and head of the Laboratory Medicine Service of the University-Hospital San Martino in Genoa, Italy. “There, they enter and alter the neurotransmitter pathways, resulting in an imbalance of neurotransmitters.”

Understanding this metabolic fingerprint could also pave the way for earlier detection of autism in children. “A newborn without any history of genetic factors for autism, such as a family history of the condition, could still be classified as at-risk by metabolomics,” Mussap said. “In the future, metabolomics could also help identify new biomarkers for use in newborn screening for autism.”

Source: News Wise

 

Blood Test: Scientists Crack Code Of Chronic Fatigue Syndrome’s Inflammatory Underpinnings

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The findings provide evidence that inflammation is a powerful driver of this mysterious condition, whose underpinnings have eluded researchers for 35 years.

The findings, described in a study published online July 31 in the Proceedings of the National Academy of Sciences, could lead to further understanding of this condition and be used to improve the diagnosis and treatment of the disorder, which has been notably difficult.

More than 1 million people in the United States suffer from chronic fatigue syndrome, also known as myalgic encephomyelitis and designated by the acronym ME/CFS. It is a disease with no known cure or even reliably effective treatments. Three of every four ME/CFS patients are women, for reasons that are not understood. It characteristically arises in two major waves: among adolescents between the ages of 15 and 20, and in adults between 30 and 35. The condition typically persists for decades.

“Chronic fatigue syndrome can turn a life of productive activity into one of dependency and desolation,” said Jose Montoya, MD, professor of infectious diseases, who is the study’s lead author. Some spontaneous recoveries occur during the first year, he said, but rarely after the condition has persisted more than five years.

The study’s senior author is Mark Davis, PhD, professor of immunology and microbiology and director of Stanford’s Institute for Immunity, Transplantation and Infection.

‘Solid basis for a diagnostic blood test’

“There’s been a great deal of controversy and confusion surrounding ME/CFS — even whether it is an actual disease,” said Davis. “Our findings show clearly that it’s an inflammatory disease and provide a solid basis for a diagnostic blood test.”

any, but not all, ME/CFS patients experience flulike symptoms common in inflammation-driven diseases, Montoya said. But because its symptoms are so diffuse —sometimes manifesting as heart problems, sometimes as mental impairment nicknamed “brain fog,” other times as indigestion, diarrhea, constipation, muscle pain, tender lymph nodes and so forth — it often goes undiagnosed, even among patients who’ve visited a half-dozen or more different specialists in an effort to determine what’s wrong with them.

Montoya, who oversees the Stanford ME/CFS Initiative, came across his first ME/CFS patient in 2004, an experience he said he’s never forgotten.

“I have seen the horrors of this disease, multiplied by hundreds of patients,” he said. “It’s been observed and talked about for 35 years now, sometimes with the onus of being described as a psychological condition. But chronic fatigue syndrome is by no means a figment of the imagination. This is real.”

Antivirals, anti-inflammatories and immune-modulating drugs have led to symptomatic improvement in some cases, Montoya said. But no single pathogenic agent that can be fingered as the ultimate ME/CFS trigger has yet been isolated, while previous efforts to identify immunological abnormalities behind the disease have met with conflicting and confusing results.

Still, the sporadic effectiveness of antiviral and anti-inflammatory drugs has spurred Montoya to undertake a systematic study to see if the inflammation that’s been a will-o’-the-wisp in those previous searches could be definitively pinned down.

To attack this problem, he called on Davis, who helped create the Human Immune Monitoring Center. Since its inception a decade ago, the center has served as an engine for large-scale, data-intensive immunological analysis of human blood and tissue samples. Directed by study co-author Holden Maecker, PhD, a professor of microbiology and immunology, the center is equipped to rapidly assess gene variations and activity levels, frequencies of numerous immune cell types, blood concentrations of scores of immune proteins, activation states of intercellular signaling models, and more on a massive scale.

Finding patterns

This approach is akin to being able to look for and find larger patterns — analogous to whole words or sentences — in order to locate a desired paragraph in a lengthy manuscript, rather than just try to locate it by counting the number of times in which the letter A appears in every paragraph.

The scientists analyzed blood samples from 192 of Montoya’s patients, as well as from 392 healthy control subjects. The average age of patients and controls was about 50. Patients’ average duration of symptoms was somewhat more than 10 years.

Importantly, the study design took into account patients’ disease severity and duration. The scientists found that some cytokine levels were lower in patients with mild forms of ME/CFS than in the control subjects, but elevated in ME/CFS patients with relatively severe manifestations. Averaging the results for patients versus controls with respect to these measures would have obscured this phenomenon, which Montoya said he thinks may reflect different genetic predispositions, among patients, to progress to mild versus severe disease.

When comparing patients versus control subjects, the researchers found that only two of the 51 cytokines they measured were different. Tumor growth factor beta was higher and resistin was lower in ME/CFS patients. However, the investigators found that the concentrations of 17 of the cytokines tracked disease severity. Thirteen of those 17 cytokines are pro-inflammatory.

TGF-beta is often thought of as an anti-inflammatory rather than a pro-inflammatory cytokine. But it’s known to take on a pro-inflammatory character in some cases, including certain cancers. ME/CFS patients have a higher than normal incidence of lymphoma, and Montoya speculated that TGF-beta’s elevation in ME/CFS patients could turn out to be a link.

One of the cytokines whose levels corresponded to disease severity, leptin, is secreted by fat tissue. Best known as a satiety reporter that tells the brain when somebody’s stomach is full, leptin is also an active pro-inflammatory substance. Generally, leptin is more abundant in women’s blood than in men’s, which could throw light on why more women than men have ME/CFS.

More generally speaking, the study’s results hold implications for the design of future studies of disease, including clinical trials testing immunomodulatory drugs’ potential as ME/CFS therapies.

“For decades, the ‘case vs. healthy controls’ study design has served well to advance our understanding of many diseases,” Montoya said. “However, it’s possible that for certain pathologies in humans, analysis by disease severity or duration would be likely to provide further insights.”

Other Stanford co-authors of the study are clinical research coordinator Jill Anderson; Tyson Holmes, PhD, senior research engineer at the Institute for Immunity, Transplantation and Infection; Yael Rosenberg-Hasson, PhD, immunoassay and technical director at the institute; Cristina Tato, PhD, MPH, research and science analyst at the institute; former study coordinator Ian Valencia; and Lily Chu, MSHS, a board member of the Stanford University ME/CFS Initiative.

The study was funded by the National Institutes of Health (grant U19AI057229), the Stanford ME/CFS Initiative Fund and an anonymous donor.

Stanford’s departments of Medicine and of Microbiology and Immunology also supported the work.

Source: scienceblog.com

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